Regulatory role of monoamine neurotransmitters in astrocytic NT-3 synthesis

Astrocytes actively control neuronal activity and synaptic transmission and by producing various neurotrophic factors represent an important local cellular source of trophic support in the normal and diseased brain. Our present study showed the ability of astrocytes to synthesize neurotrophin-3 (NT-3) and the active involvement of the monoamine neurotransmitters noradrenaline, adrenaline, dopamine, and serotonin, as well as basic intracellular second messenger systems, in the regulation of NT-3 production in neonatal rat cortical astrocytes. Using a new NT-3 specific enzyme-immunoassay, we showed that neonatal rat cortical and, for comparison, cerebellar astrocytes in primary culture can synthesize NT-3; the basal cellular content of NT-3 protein was 23.2+/-0.4 pg NT-3/mg cell protein and 23.6+/-0.9 pg NT-3/mg cell protein, respectively. The examined neurotransmitters, with the exception of serotonin, were able to potently and transiently increase NT-3 mRNA and NT-3 protein content; their maximal effects were dose- and time-dependent. Noradrenaline (1 microM), adrenaline (1 microM), and dopamine (100 microM) showed a maximal increase in NT-3 cellular content after 6h treatment causing a 1.9-, 1.8- and 2.7-fold elevation, respectively. Prior to the observed increase in NT-3 protein levels, the examined catecholamines increased NT-3 mRNA levels with maximal effects observed after 1h (noradrenaline) and 2h (adrenaline and dopamine) of incubation causing 2.4-, 2.6- and 3-fold elevation, respectively. Screening different activators of basic intracellular second messenger systems for their influence on NT-3 synthesis revealed that forskolin (20 microM), dibutyryl cAMP (dBcAMP) (100 microM), as well as calcimycin (1 microM) (Ca(2+) ionophore A23187) and phorbol 12-myristate 13-acetate (TPA) (100 nM), markedly increased the cellular level of NT-3 protein. Neurotransmitter-induced NT-3 levels were susceptible (to varying degrees) to inhibition by H-89 (protein kinase A inhibitor) or staurosporin (protein kinase C inhibitor), which led us to conclude that downstream signaling responsible for the stimulation of NT-3 synthesis by monoamines in astrocytes consists of multiple, complex intracellular mechanisms involving the cAMP/protein kinase A pathway, activation of protein kinase C, as well as mobilization of Ca(2+) ions. Our results indicate for the first time that monoaminergic neurotransmitters play an important role in the regulation of NT-3 synthesis in cultured rat astrocytes.